RESUMO
Counterion atmospheres condensed onto charged biopolymers strongly affect their physical properties and biological functions, but have been difficult to quantify experimentally. Here, monovalent and divalent counterion atmospheres around DNA double helices in solution are probed using small-angle x-ray scattering techniques. Modulation of the ion scattering factors by anomalous (resonant) x-ray scattering and by interchanging ion identities yields direct measurements of the scattering signal due to the spatial correlation of surrounding ions to the DNA. The quality of the data permit, for the first time, quantitative tests of extended counterion distributions calculated from atomic-scale models of biologically relevant molecules.
Assuntos
DNA/química , Cátions , Metais/química , Conformação de Ácido Nucleico , Espalhamento de Radiação , Soluções , Eletricidade Estática , Raios XRESUMO
We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/metabolismo , Tetrahymena/genética , Animais , Sequência de Bases , Cálcio/farmacologia , Cristalografia por Raios X , Radical Hidroxila/metabolismo , Cinética , Magnésio/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Catalítico/genética , Sódio/farmacologia , Eletricidade Estática , Tetrahymena/enzimologiaRESUMO
Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.
Assuntos
Precursores de Proteínas/química , Timosina/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Cátions Bivalentes/farmacologia , Dicroísmo Circular , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Timosina/química , Timosina/genética , Timosina/metabolismo , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.
Assuntos
Dobramento de Proteína , Precursores de Proteínas/química , Timosina/análogos & derivados , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas Recombinantes/química , Soluções , Timosina/químicaRESUMO
We have directly characterized the extent of chain collapse early in the folding of protein L using time-resolved small angle X-ray scattering. We find that, immediately after the initiation of refolding, the protein exhibits dimensions indistinguishable from those observed under highly denaturing, equilibrium conditions and that this expanded initial state collapses with the same rate as that of the overall folding reaction. The observation that chain compaction need not significantly precede the rate-limiting step of folding demonstrates that rapid chain collapse is not an obligatory feature of protein folding reactions.
